Rpmi 1640 culture medium

SK-MEL-2 cells containing the Luc gene (JCRB cell bank) were maintained at 37 °C in a humidified 5% CO₂ incubator using RPMI1640 culture medium (FUJIFILM Wako Pure Chemical) containing 10% heat-inactivated fetal calf serum (Sigma–Aldrich), 100 U/ml penicillin, and 100 μl/ml streptomycin (Nacalai Tesque) in 10 cm dish (Thermo Fisher Scientific). Dulbecco’s modified Eagle’s medium (FUJIFILM Wako Pure Chemical) or Iscove’s modified Dulbecco’s medium (Nacalai Tesque) was used to maintain A549 or HAP1 cells as previously described (16 (link), 25 (link)). Zinc-supplementation experiments involved adding the indicated concentrations of ZnSO₄ (Nacalai Tesque) to the cell culture medium.

DC2.4 cells, an immature DC cell line, were cultured in RPMI 1640 culture medium (Wako) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 0.05 mM 2-mercaptoethanol, 10 mM HEPES, 1 mM sodium pyruvate, 100 μM minimal essential medium (MEM) non-essential amino acids, 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Wako). RAW 264.7 cells, a mouse macrophage cell line, were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 μM MEM non-essential amino acids (Sigma–Aldrich), 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Sigma–Aldrich). DC2.4 cells (5 × 10⁴ cells) and RAW 264.7 cells (5 × 10⁴ cells) were seeded in 96-well culture plates, respectively, and incubated to adhere overnight at 37°C in a humidified atmosphere of 5% CO₂. After the incubation, the cells were treated with 10 μg/mL LH2171 and incubated for a further 4 h. Then, DC2.4 cells were stimulated with LPS from Escherichia coli 055:H5 (Sigma–Aldrich) at a final concentration of 1 μg/mL, and RAW 264.7 cells were stimulated with LPS from E. coli 0111:B4 (Sigma–Aldrich) at a final concentration of 10 μg/mL for 20 h. In all experiments, the cells were grown to 80–90% confluence.